Our finding that AQP2 constitutively recycles in the absence of the antidiuretic hormone, vasopressin (VP), has led to a rethinking of how VP causes AQP2 membrane accumulation and urinary concentration. Indeed, we recently found that an FDA approved drug, simvastatin, blocks AQP2 endocytosis and reduces urine output in VP- deficient Brattleboro rats. Our proposed studies continue the strategy of combining cell biological interrogation of AQP2 trafficking with functional studies in vivo to generate novel therapeutic approaches for disorders of fluid balance including NDI, as well as congestive heart failure and hyponatremia. In Aim 1, we will explore the mechanism(s) underlying our recent discoveries that AQP2 itself is a catalyst for VP-induced actin depolymerization and that the actin-binding protein ezrin is an AQP2 associated protein involved in AQP2 endocytosis. Agents that target the ezrin binding domain on AQP2 would provide increased specificity of action for NDI treatment. Aim 2 will first expand our promising work on phosphodiesterase (PDE5) inhibitors, calcitonin and statins, which promote AQP2 membrane accumulation and increase urine concentration. We will optimize treatment doses, times and delivery routes, and assess the effectiveness of drug combinations. Then, we will further develop our new chemical screening program to identify and characterize compounds that modulate exocytosis and endocytosis in AQP2- expressing cells using high throughput, fluorescence-based assays in cell cultures (we have already identified 40 chemicals that need further characterization). Compounds that modify (increase or decrease) AQP2 membrane accumulation in vitro will ultimately be tested in vivo. We will use a multidisciplinary approach ranging from in vitro protei association assays, novel cell culture models, cellular imaging and analysis (using the PMB Microscopy Core), high throughout chemical screening (via the Center for Systems Biology Chemical Biology COre) and whole animal studies. Our overall goal is to design novel therapies for water balance disorders, including NDI and hyponatremia, using a combination of directed and discovery approaches that bypass the VP/V2R signaling cascade to modulate cell surface expression of AQP2.